In situ monitoring of Lentilactobacillus parabuchneri biofilm formation via real-time infrared spectroscopy

Foodborne pathogenic microorganisms form biofilms at abiotic surfaces, which is a particular challenge in food processing industries. The complexity of biofilm formation requires a fundamental understanding on the involved molecular mechanisms, which may then lead to efficient prevention strategies. In the present study, biogenic amine producing bacteria, i.e., Lentilactobacillus parabuchneri DSM 5987 strain isolated from cheese were studied in respect with biofilm formation, which is of substantial relevance given their contribution to the presence of histamine in dairy products. While scanning electron microscopy was used to investigate biofilm adhesion at stainless steel surfaces, in situ infrared attenuated total reflection spectroscopy (IR-ATR) using a custom flow-through assembly was used for real-time and non-destructive observations of biofilm formation during a period of several days. The spectral window of 1700–600 cm−1 provides access to vibrational signatures characteristic for identifying and tracking L. parabuchneri biofilm formation and maturation. Especially, the amide I and II bands, lactic acid produced as the biofilm matures, and a pronounced increase of bands characteristic for extracellular polymeric substances (EPS) provide molecular insight into biofilm formation, maturation, and changes in biofilm architecture. Finally, multivariate data evaluation strategies were applied facilitating the unambiguous classification of the observed biofilm changes via IR spectroscopic data.


Supplementary note 1 Experimental strategy for L. parabuchneri cultivation
Lactic acid bacteria are Gram positive aerotolerant microorganisms that synthesize lactic acid as the main product of sugar fermentation. The culture media and the main fermentation conditions requires a nutritive media rich with carbon source 1 . Mann de Rogosa Sharpe broth has been used as an oxygen free media for cultivation of the L. parabuchneri DSM 5987 isolates. To test the capacity of strain for Histamine producing, the cultures have been supplemented with 5mM Histidine 2 . By the scheme you can see the matrix of the overnight inoculated samples incubated on 30°C, 24-72h. After that the supernatant was discarded and resuspended on fresh MRS media, stirred for 2 h and afterwards measuring the OD. Before the inoculation to the flow system, the media was flushed by nitrogen gas (degassing process) and the oxygen level were monitored using an O 2 microsensor.

Supplementary note 2
The process of L. parabuchneri biofilm formation at the ATR waveguide surface with five distinct stages of biofilm evolution Development of lactobacillus biofilms leads toward series of morphological changes on the substrate 3,4 . The adhesion to surface is dependent physicochemical properties of the abiotic and biotic surfaces like: pH, hydrophobicity, surface texture, temperature, etc 5 . The five proposed stages of biofilm formation are: 1) initial attachment of cells at the surface; 2) transport and attachment of bacterial cells towards the surface; 3) early development of biofilms indicated by EPS production and proliferation into microcolony units; 4) maturation of the biofilm architecture, and 5) dispersal of mature clusters and cell detachment (schematically illustrated in Figure 2). Many cells can detach from the surface and come back to the planktonic lifestyle, spreading out to start a new cycle of life. Extracellular polymeric matrix has the key role to adhesion ability and strength the bond between bacteria and the substrate.

Starting a new cycle
Supplementary note 3

IR-spectroscopic characterization of the individual components of de Man Rogosa and Sharpe medium (MRS)
IR-ATR spectroscopy with the Alpha I spectrometer of the individual constituents of MRS showed the characteristic spectra in the spectral region from 1800 -900 cm -1 . The peaks of the individual constituents were integrated and averaged thereafter served as basis for the cumulative impulse fit demonstrated in the manuscript. Spectra were recorded using the same Alpha spectrometer with Platinum Single bounced Diamond Crystal in 2 cm -1 resolution and 100 average scans. The characterization of the MRS growth medium is crucial to chemically analyze L. parab. adsorption and biofilm formation in molecular detail and the cumulative impulse fit of the spectra of de Man Rogosa and Sharpe medium (MRS) is fundamental for the analysis of L. parab. biofilms in this culture medium.

Supplementary discussion
Optical microscopy of L. parabuchneri biofilms The characterization of the chemistry and architecture of biofilms can be evaluated by the combination of IR-ATR spectroscopy and light microscopy and therefore achieve the information on the region which is restricted by evanescent wave depth penetration 6 . For 7 days inoculation on the flow ATR system, the ZnSe surface has become highly colonized by L. parabuchneri bacteria creating high density biofilms with different heterogeneity types. These images show uniform coverage of ZnSe surface by L. parabucneri biofilms. Figure  5 a show the biofilms visualized after 24 h (1 day), figure 5 b shows the biofilm attached to waveguide surface after 72 h (3 days) and figure 5 c shows the mature biofilm after 168 h (7 days). For 7 days of inoculation within the IR-ATR assembly, the ZnSe surface has become highly colonized by L. parabuchneri bacteria creating high-density biofilms with spatially different heterogeneities. Optical microscopy is a basic technique to track the structural heterogeneity of biofilms. The optical micrographs prove the coverage with biofilms at the waveguide surface. These images reveal biofilm structures adherent to the ZnSe surface in small colonies of cells after 24 h (Fig 5 a), whereby the number of L. parabuchneri cells that were near the attached 'pioneering cells' was higher than those observed close to the initially uncovered zones of the ZnSe substrate. A uniform coverage of the ZnSe surface by L. parabuchneri biofilms was observed after 3 days (Fig 5.b) of real-time monitoring with the addition of fresh nutritive media every 16 h. The aged biofilms after 7 days (Fig 5.c) reveal an increased thickness of the biofilm enriched with nutrients for a week. Microscopic images were captured with Zeiss Microscope (Zeiss, Germany) and defined by the image analysis micro software.  υC-C, υP-O-P RNA backbone Ribosomes 900-500 -"true" fingerprint region Bands not assigned to specific functional groups